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The Use of Microbial Community Fingerprinting As a Marker for Tracking the Source of Water: Application to Pathogen and Groundwater Source Tracking

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The Use of Microbial Community Fingerprinting As a Marker for Tracking the Source of Water: Application to Pathogen and Groundwater Source Tracking Graduate Theses, Dissertations, and Problem Reports 2010 The use of microbial community fingerprinting as a marker for tracking the source of water: Application to pathogen and groundwater source tracking Mary Clement West Virginia University Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Clement, Mary, "The use of microbial community fingerprinting as a marker for tracking the source of water: Application to pathogen and groundwater source tracking" (2010). Graduate Theses, Dissertations, and Problem Reports. 3210. https://researchrepository.wvu.edu/etd/3210 This Thesis is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Thesis in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Thesis has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. The use of microbial community fingerprinting as a marker for tracking the source of water: Application to pathogen and groundwater source tracking Mary Clement Thesis submitted to the College of Engineering and Mineral Resources at West Virginia University in partial fulfillment of the requirements for the degree of Master of Science in Civil and Environmental Engineering Approved by: Benoit Van Aken, Ph.D., Committee Chairperson Radhey S. Sharma, Ph.D. Lian-Shin Lin, Ph.D. Dorothy Vesper, Ph.D. Department of Civil and Environmental Engineering Morgantown, West Virginia 2010 Key words: Terminal-restriction fragment length polymorphism, microbial community fingerprinting, microbial source tracking, genomic cloning library Abstract The use of microbial community fingerprinting (using T-RFLP) as a marker for tracking source of water: Application to pathogen and groundwater source tracking Mary Clement Microbial community structure in groundwater is extremely complex and constitutes a fingerprint of water. Terminal restriction fragment length polymorphism (T-RFLP) analysis is a polymerase chain reaction (PCR)-based fingerprinting method that is commonly used for comparative microbial community analysis, and to track specific bacterial markers in complex environments. The Objective of this research was to propose an innovative approach for tracking the origin of groundwater, microbial source tracking and origin of surface water, based on the specific structure of the microbial community using T-RFLP analysis, which can be used for microbial source tracking. The first specific aim was to validate the use of T-RFLP profiles of deep and shallow waters in Huntingdon, PA, Great Valley, WV and Berkeley springs, WV, as a marker of the groundwater source. Alternatively, a 16S ribosomal DNA genomic library was constructed to identify bacterial species that can inform about the source of groundwater. It was observed that clustering based on screening a clone library gave more reliable information of the groundwater source than the T-RFLP profiles. The second specific aim focused on the characterization of the microbial community from different streams along the Left Fork Mud River watershed in Lincoln County, WV. T-RFLP analyses were carried out for determining the source of contaminated water. Results showed that different streams of the Mud River watershed has a specific microbial terminal restriction fragment (TRF) profiles, which helps in determining the origin of water. The third specific aim was to characterize the entire microbial community in several streams in Corridor-H watersheds, WV using T-RFLP analysis. It was observed that water samples collected from different locations of the same stream carried a very specific signature that can be used for microbial source tracking analysis. The T-RFLP method application on spring waters and Mud River watersheds was based on the preliminary results obtained from the analysis of the microbial community structure of several streams in Corridor-H watersheds. Our results, thus provide the evidence that natural waters has a „microbial signature‟ (T-RFLP profile) that can be used as a marker for pathogens and groundwater source tracking. Acknowledgements There are a number of people I would like to sincerely thank for the guidance, assistance, and insightfulness they provided to allow me to complete this research and thesis. Foremost, I would like to thank my advisor and thesis director, Dr. Benoit Van Aken, for his valuable guidance, advice and support. I also wish to thank my committee members: Dr. Radhey Sharma, Dr. Lian- Shin Lin, and Dr. Dorothy Vesper, for their helpful comments and suggestions. Having a chemical engineering background and performing a molecular biology-based research project resulted in having to learn a substantial amount of concepts and information that were previously unknown to me. I would like to thank Dr. Harris Edenborn (NETL), Dr. Dorothy Vesper (Committee member) and T. Vandivort from WVWRI for providing with data, which has been used in this thesis. Very special thanks to my lab mates Melin and Paola, for their valuable help and suggestions in learning laboratory techniques. I would like to thank Danielle Ellis and Dhiraj from Biology department at WVU for T-RFLP technology support. I sincerely express my gratitude towards everyone mentioned in these acknowledgements! Thank you all! Mary Clement iii Table of Contents List of symbols / Nomenclature ..................................................................................................... ix Chapter 1: General Introduction ..................................................................................................... 1 1.1 Introduction ........................................................................................................................... 1 1.2 Statement of critical regional or state water problem ........................................................... 2 1.3 Objective of the Research ..................................................................................................... 6 1.4 Working Hypothesis ............................................................................................................. 6 1.5 Significance........................................................................................................................... 6 1.6 Specific aims ......................................................................................................................... 6 2.1 Microbial communities in natural water ............................................................................... 8 2.1.1 Microbial ecology in natural waters .............................................................................. 8 2.1.2 Bioindicators for water quality .................................................................................... 10 2.2 Microbial Source Tracking ................................................................................................. 10 2.3 Molecular DNA fingerprinting methods ............................................................................. 12 2.4 Terminal-restriction fragment length polymorphism analysis (T-RFLP) ........................... 13 2.5.1 Disadvantages of the T-RFLP Method ........................................................................ 14 Chapter 3: Materials and Methods ................................................................................................ 15 3.1 Water samples ..................................................................................................................... 15 3.1.1 Spring water ................................................................................................................. 15 3.1.2 Stream water ................................................................................................................ 16 3.2 Membrane filtration bacterial enumeration ........................................................................ 17 3.3 DNA Extraction .................................................................................................................. 17 3.4 Microbial community fingerprinting using T-RFLP .......................................................... 18 3.4.1 16S rDNA Amplification ............................................................................................. 19 3.4.2 Agarose Gel Electrophoresis (AGE) ............................................................................ 19 3.4.3 Enzymatic digestion ..................................................................................................... 20 3.4.4 Capillary electrophoresis ............................................................................................. 20 3.4.5 Terminal fragment profile analysis .............................................................................. 21 3.5 Bacterial Identification by screening of 16S rDNA gene library ....................................... 22 Chapter 4: Results and discussion................................................................................................. 24 4.1 Specific aim 1 ....................................................................................................................
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